For functional studies, it is crucial to analyze individual regions of a protein systematically. This is usually performed by randomizing all amino acids of the region or by varying few conserved positions depending on the size of the region of interest. On one hand, such data results in the identification of key amino acids determining the interaction of a protein region for example with another protein region, with DNA, or with lipid structures. On the other hand, expression in a cellular environment of either a randomized protein region alone or of a randomized region in the context of a full protein directly contributes to the understanding of the protein's function. Another application of region randomization is changing the binding properties of a single chain antibody (scFv). A scFv consists of variable regions of both the light and heavy chain. Each chain features three variable regions, called CDR1, CDR2, and CDR3. Of the six CDRs, the CDR3 of the heavy chain is the major determinant of the scFv's affinity for its antigen. For changing the binding properties of a scFv, the heavy chain CDR3 region of a scFv with a given interaction profile is randomized and screened against the epitope of interest. This usually leads to the identification of scFvs with a low affinity. For improving the affinity, the light chain CDR3 of the novel scFv is then randomized. Therefore, stepwise randomization of two scFv variable regions can be used to isolate new or better binders against a given epitope.
The classical protocol for region replacement/randomization in the context of library production is performed usually in bacteria such as E. coli. It involves production of PCR fragments representing the library of regions, digestion with restriction enzymes cutting at or close to the end of the fragments of interest, and ligation into a vector linearized with matching restriction enzymes.
For use in yeast, libraries have been produced directly in yeast with homologous recombination (Hua, Luo et al. 1998). The process requires production of a donor DNA fragment bearing on each end homologies to the linearized target vector. Fragments for use in homologous recombination can therefore be produced by PCR with primers bearing the homologous sequences at their 5′ ends.
US patent application published under No. US2002/012734 describes a method for directed evolution, gene reassembly and directed mutagenesis using homologous recombination and selectable markers for counterselection in bacteria.
Although there exist already methods for the construction of randomized gene sequence libraries by means of homologous recombination in cells, there is still a need for methods which allow an efficient construction and selection of such randomized libraries.